Methotrexate (MTX) is used in the treatment of rheumatoid arthritis (RA) due to its anti-inflammatory properties. However, its ability to suppress macrophage derived proinflammatory cytokines has not been explained. The aim of this study was to compare the therapeutic effects of high-dose MTX on the development of collagen-induced arthritis (CIA) High-dose MTX reduced significantly both the incidence/severity of CIA and the serum levels of IgG anti-CII. Surprisingly, macrophages obtained from MTX-treated mice and stimulated in vitro with LPS produced more TNF-, IL-6 and IL-12p40 than the control cells. MTX added in vitro to peritoneal macrophages did not affect the cytokine production. However, incubation of proliferating RAW 264.7 macrophages with MTX resulted in severe suppression of all proinflammatory cytokines tested. In conclusion MTX treatment markedly ameliorates arthritis while it does not suppress the cytokine release from peritoneal macrophages. In vitro study indicates that MTX can inhibit the cytokine production if target cells proliferate.